The long-term focus of the research in this proposal is to understand the role of lipid membrane composition on multivalent ligand/receptor binding. Recent developments with model bilayer systems have shown that structured liquid-ordered regions rich in cholesterol and sphingolipids can coexist along side a phospholipid rich, liquid-disordered domain. It is still controversial as to whether these "lipid rafts" also exist in cells; however, there has been speculation that if they do exist in vivo, they could significantly enhance multivalent ligand-receptor attachment. The mechanism might involve either concentrating membrane bound ligands into small highly concentrated regions or changing the ligand orientation to make binding with external proteins more favorable. The hypothesis that membrane composition and orientation can directly affect the equilibrium dissociation constants of multivalent binding will be tested for IgG antibodies (a bivalent system) and for cholera toxin (a pentavalent system). Both initial binding as well as subsequent lateral binding will be explored using newly developed high-throughput microfluidic strategies. Such fundamental studies may ultimately play a role in developing inhibitors to the initial step of pathogen entry into cells. The four specific aims of this proposal include: (1) investigating the initial binding event for anti-2,4 DNP IgG with its lipid conjugated hapten. Studies will be conducted as a function of cholesterol content as this may force the ligand out of the membrane; hence, making it more available. (2) Investigations of the second dissociation constant for antibodies will be conducted. This will be done as a function of cholesterol content, ligand density, and sites of unsaturation in the lipid tails. One central hypothesis is that components which attenuate the diffusion constant of the membrane may also impede lateral binding interactions. (3) The effect of lipid raft formation on binding will be undertaken. Mixtures of cholesterol/sphingolipid/phospholipid will be probed for their ability to affect both the initial and subsequent binding interactions in membranes. Measurements of binding will be made in both the phospholipid-rich and sphingolipid-rich phases when possible. (4) The above three aims will be expanded to cholera toxin/GM1. This system should show dramatically different behavior because GM1 is known to form clusters, rather than acting as an ideal dilute constituent of the membrane. The binding constant may change dramatically above a critical concentration.